Abstract

Technical advancements in research techniques in science are made in slow increments. Even so, large advances from insight and hard work of an individual with a single technique can have astonishing ramifications. Here, we examine the impact of Dr. Maurice B. Burg and the isolated perfused renal tubule technique and celebrate the 50th anniversary of the publication by Dr. Burg and his colleagues of their landmark paper in the American Journal of Physiology in 1966. In this study, we have taken a scientific visualization approach to study the scientific contributions of Dr. Burg and the isolated perfused tubule preparation as determining research impact by the number of research students, postdoctoral fellows, visiting scientists, and national and international collaborators. Additionally, we have examined the research collaborations (first and second generation scientists), established the migrational visualization of the first generation scientists who worked directly with Dr. Burg, quantified the metrics indices, identified and quantified the network of coauthorship of the first generation scientists with their second generation links, and determined the citations analyses of outputs of Dr. Burg and/or his first generation collaborators as coauthors. We also review the major advances in kidney physiology that have been made with the isolated perfused tubule technique. Finally, we are all waiting for the discoveries that the isolated perfused preparation technique will bring during the next 50 years.

isolated tubules

nephron segments

perfused tubule preparation

ion transport

scientific visualization

research network

technical developments in research techniques in science are made in slow increments. Even so, large advances from insight and hard work of an individual with a single technique can have astounding ramifications. How does one quantify the contribution of a single scientist and/or his/her research technique? When one decides whom to research, one certainly has to think of a scientist whose technique has spanned a generation(s) of researchers and who has led the way from basic science into the “molecular age of science.” When one thinks of major technical contributions to science, as an epithelial ion transport physiologist I (K. L. Hamilton) would certainly identify Hans H. Ussing for the Ussing chamber and the measurement of short-circuit current (30, 49); Bert Sackmann and Edwin Neher who shared the Nobel Prize in Physiology or Medicine in 1991 for the patch-clamp technique (40); and Kary B. Mullis and Michael Smith who shared the Nobel Prize in Chemistry in 1993 for Mullis' work on PCR and Smith's work on site-directed mutagenesis (25, 39).

If one thinks of a person who stands out in the field of renal physiology, in the second half of the 20th century, one immediately thinks of Dr. Maurice (Moe) B. Burg. In this paper, we examine the impact of Dr. Burg, who with his colleagues published the isolated perfused renal tubule technique in 1966 in the American Journal of Physiology (6). Dr. Burg (Fig. 1) is highlighted here in honor of the 50th anniversary of the development of the isolated perfused renal tubule technique (6), which revolutionized scientists' ability to conduct research on the various segments of the nephron, the functional unit of the kidney. Dr. Jared J. Grantham (Dr. Burg's second research fellow) summed up his thoughts of the impact of the isolated tubule technique as follows: “The isolated perfused tubule opened the mysterious black box we call the kidney to direct analysis” (17) (Grantham JJ, personal communication). Numerous scientists have trained directly with Dr. Burg at his laboratory in the National Institutes of Health (NIH; Bethesda, MD) or with those first generation scientists (research fellows, visiting scientists) to use the isolated perfused tubule technique. Other scientists have established the isolated perfused renal tubule preparation or other perfused structures (e.g., pancreatic duct, rectal gland, gastric duct, colonic crypt, sweat duct, Malpighian tubule) independently of Dr. Burg. It should be noted that Dr. William H. Dantzler (Univ. of Arizona) developed his own version of the isolated perfusion tubule system and had read the 1966 paper (Dantzler WH, personal communication). In 1973, Dr. Dantzler's first perfused tubule paper appeared, in which he examined urate transport of the snake proximal tubule (11). He published many studies on various functions of isolated perfused tubules from amphibian, reptilian, avian, and mammalian species. There were/are also a number of “hubs” of perfused isolated tubule research based in Germany, which included work by Drs. Rainer Greger, Karl J. Ullrich, Florian Lang, Gerhard Rumich, and Hans Oberleithner, among others. It should be noted that Dr. Greger (with the support of Dr. Ullrich and the resources of the Max Planck mechanical workshops in Frankfurt) drastically revised the concentric pipette assemblies to be more suitable for transepithelial electrophysiology, and he really established intracellular electrophysiological analyses in the isolated tubule (Schafer JA, personal communication). Collectively, the research outputs from the use of Dr. Burg's technique has led the way to major discoveries that furthered understanding of the functions of the various segments of the nephron (discussed later). Dr. Burg originally isolated tubules from the rabbit; however, scientists have since used other tissues from other species, including humans, mice, flies, salamanders, flounders, rats, frogs, sharks, birds, mosquitoes, and hamsters, for example.

Brief Background of Dr. Burg

Dr. Burg was born in Boston, MA, on April 9, 1931. He graduated with an AB cum laude from Harvard College in Cambridge, MA, in 1952. This degree was followed in 1955 with his MD from Harvard Medical School. Next, he completed an internship at the Beth Israel Hospital in Boston followed, by a residency in medicine at the Boston Veterans Administration (VA) Hospital. He then moved to the National Institutes of Health (NIH; 1957–1959) and was mentored by Dr. Jack Orloff [future Scientific Director of the National Heart, Lung and Blood Institutes (NHLBI) from 1974–1988] (13); followed by an additional year at the Boston VA. Dr. Burg returned again to the NIH in 1960 as an Investigator in the Laboratory of Kidney and Electrolyte Metabolism (LKEM) at the NHLBI. He was the Chief of the LKEM from 1975 until 2002, and since then he has been the Chief of the Renal Cellular and Molecular Biology Section of the LKEM (Burg MB, personal communication). Finally, Dr. Burg became Scientist Emeritus of the NHLBI in 2013; and he still maintains a working relationship with the kidney research laboratory at the NIH (www.nhlbi.nih.gov/research/intramural/researchers/pi/burg-maurice).

This contribution is not meant to be a “true” biography of Dr. Burg; however, we will examine the overall research impact of Dr. Burg's isolated perfused tubule preparation. Interested readers are directed to the American Physiological Society's web page Living History of Physiology (http://www.the-aps.org/mm/Membership/Living-History/Burg) and the ISN Video Legacy Project (cyber nephrology.ualberta.ca/ISN/VLP/Trans/burg.htm, ISN, 26) for interviews with Dr. Burg; which include highlights of his career. These web pages were the source for some of the material presented in this section and personal information provided by Dr. Burg.

Pre-Isolated Perfused Tubule Era: The Renal Micropuncture Technique

Before the development of the isolated perfused tubule preparation, the renal micropuncture technique was, and still is, a formidable renal technique that ushered in the age of experimentation of the nephron. Indeed, in the 1920s Wearn and Richards (53) developed and established the micropuncture technique in the frog that allowed them the ability to puncture nephrons to determine the composition of the ultrafiltrate. Additionally, Wearn and Richards reported that the concentration of Cl−, K+, glucose, urea, and the pH of the glomerular filtrate were very similar to that of plasma. Lorenz (35) and others (45, 50) remarked that Wearn and Richards (53) provided the first experimental evidence that the glomerular filtrate was protein free and that the nephron exhibited tubular reabsorption. Subsequently, Sands (45) stated that “The development of the micropuncture by Wearn and Richards in 1924 ranks as one of the greatest advances in renal physiology during the 20th century. . . .” The quotation by Sands continued with “. . . .along with the development of the isolated perfused tubule by Burg and colleagues in 1966.” Some have noted that the resurgence in the use of the micropuncture technique, in light of the gene-targeted mouse era, is overshadowed by the dwindling number of physiologists being trained in this essential renal physiological technique (45, 50). The same comment is true for the isolated perfused tubule technique. For further historical perspective and technical details of the micropuncture technique, the reader is directed to the following excellent reviews (34, 35, 50).

Development of the Isolated Perfused Tubule Preparation

During his first time at NIH, Dr. Burg noted that the prominent method for studying kidney function was by clearance measurements (3). Although the micropuncture technique was a powerful renal tool, Dr. Burg believed the general method was relatively underutilized (3). He envisioned a different version of micropuncture that would allow scientists to investigate the functional properties of individual segments of the nephron (3). While at the NIH, Dr. Burg became intrigued with the idea of perfusing renal tubules outside of their normal environment in the kidney (Grantham JJ, personal communication). His idea was based on two important series of experiments. First, Dr. Hans H. Ussing of Denmark conducted his exquisite experiments examining the ion transport properties of epithelial sheets (frog skin) isolated between two chambers containing bathing solution (30, 49). Thus Ussing examined transcellular function of an epithelial tissue in vitro with his chamber and the short-circuit technique. Second, Dr. Burg was well aware of the advances that neuroscientists were making with new methods to describe the action potential of the giant squid axon (24). The ideas and technical advances made to examine the properties of epithelial tissues and the axon lead Burg to write “While these were single cells, not tubes of epithelia, and while they were much larger than kidney tubules the successes with the giant axons were well known by the late 1950s and suggested to me that in vitro perfusion might be an experimental approach to kidney tubules” (3).

By the time Dr. Burg returned to the LKEM in 1960, others had established the micropuncture technique in the laboratory, so he decided to concentrate his efforts on his idea of perfusing the renal tubule (3). We will not attempt here to provide the reader with an extensive review of Dr. Burg's development of the isolated perfusion technique (6), but we direct the reader to reviews written by Dr. Burg for specific details (2, 3, 7, 8).

Here, we will briefly describe the major developments of the isolated perfused tubule apparatus. Dr. Burg and his group were faced with three immediate goals 1) learning how to isolate specific segments of the nephron, 2) how to mount the isolated tubule within a perfusion system, and 3) how to collect fluid from the distal end of the perfused tubule. Dr. Burg started the quest to isolate segments of the rabbit nephron with a collagenase approach that was a commonly used method to isolate cells at that time. However, the collagenase approach was appropriate for obtaining a suspension of tubules, but was not as effective for isolating single tubules, because the tubules were too fragile (3, 6). However, in 1963, a visit by the renal anatomist Dr. Ivar Sperber to the LKEM changed Dr. Burg's “research luck.” Dr. Sperber taught Dr. Burg how to hand-microdissect individual nephrons with forceps rather than micromanipulators (3). Next, his first fellow Dr. Maurice Abramow, joined the laboratory in 1963, followed by Dr. Jared J. Grantham in 1964. Dr. Grantham made an immediate impact by dissecting rabbit tubules without collagenase and using Sylgard to electrically seal perfused tubules (Grantham JJ, personal communication). The next technical step was to determine how to mount the isolated tubule in a chamber between holding pipettes to allow perfusion of the tubule lumen. Dr. Burg began to construct glass micropipettes in an effort to hold the isolated tubule on one end with one pipette while trying to apply suction to the opposite end with another pipette (3). However, he realized that this approach was not a suitable way of securing the tubule. In due time, Dr. Burg thought that concentric pipettes (Fig. 2) might be the answer as he could apply suction all around the tubule; therefore, the outer pipette was used for suction while the inner pipette was used to cannulate the tubule (3, 26). The next piece of the “perfusion puzzle” was having a concentric pipette system that allowed axial positioning of the micropipettes with respect to each other. For this, Dr. Burg visited with Dr. Peter W. Davies (at Johns Hopkins University), who had developed an apparatus that held and moved micropipettes precisely as Dr. Burg desired; however, Dr. Davies' delicate apparatus had to be reassembled each time it was needed for an experiment (3). Therefore, when Dr. Burg returned to NIH, he met with Kenneth Bolen, head of the precision machining unit, and Jim White, and they designed a more robust pipette-holding apparatus that is essentially the same still used today (Fig. 3). Finally, Dr. Grantham solved the last technical aspect of the technique, designing and developing the pipette used to collect fluid from the distal end of the perfused tubule. Indeed, Dr. Burg stated “. . . .If anyone is responsible for the progress in that field, it's Jared . . . he . . . had one pipette that he sucked the tubule into, had oil in the pipette, and introduced a collection pipette to collect the fluid that had accumulated” (26).

Procedure for tubule perfusion. Concentric pipettes are on the left. The end of the tubule is drawn by suction into the tip of the outer pipette, which supports it and seals the inner pipette within the tubule lumen. Collecting pipettes are on the right. The tubule is drawn into the tip of the outer collecting pipette by suction and remains lodged there when the suction is stopped. The inner pipette is introduced periodically to remove aliquots of fluid. (The figure is the original Fig. 2 and the original legend from Ref. 6).

Apparatus for perfusing isolated tubule fragments. (The figure is the original Fig. 1 from Ref. 6).

Over the subsequent years, modifications to the perfusion system have occurred. Dr. Mark A. Knepper (a fellow with Dr. Burg) reminded the authors of the contributions of Gerald G. Vurek and Robert L. Bowman of the LKEM to the success of the isolated perfused tubule. Dr. Knepper stated that they “produced multiple devices that allowed us to make measurements in the 5–100 nanoliter samples collected from the perfused tubules. The earliest ones that may be familiar to readers are the helium glow photometer (Bowman) and the picapnotherm (Vurek). Bowman and Vurek could be considered major players in the success of the isolated perfused tubule” (Knepper MA, personal communication). One such device is seen in the background of Moe's picture in Fig. 1.

Of course, all of the work by Drs. Burg, Grantham, Abramow, and Orloff culminated in the classic 1966 paper (6). A perfused collecting tubule from the original paper is shown in Fig. 4 (6). Figure 5 shows the isolated perfused tubule rig that Dr. Burg gave to Dr. Knepper when Dr. Knepper started his urea studies (Knepper MA, personal communication).

Photo of a isolated perfused tubule rig. Perfusion is from right to left in this rig. This is the rig that Dr. Moe Burg gave to Dr. Mark A. Knepper for his original urea studies. (Photo courtesy of Dr. Mark A. Knepper, circ. 1980).

Readers are directed to Burg and colleagues (7), in which the original Burg et al. (6) paper was reprinted with commentaries by Drs. Burg and James A. Schafer (a visitor to the Burg laboratory). Dr. Grantham has summarized his experience and contributions to the development of the isolated perfused tubule technique in his autobiography (17). Additionally, for interested readers, there was a dedicated issue of Kidney International (volume 22 Issue 5, Nov 1982) devoted to the advances of the perfused tubule preparation (http://www.sciencedirect.com/science/journal/00852538/22/5).

Research Collaborations, Migrational Visualization, and Metrics Indices: Research Impact of Dr. Burg

Of course, the significance of a scientific contribution can be examined in various ways (for this paper, we are using scientific visualization methods) such as determining research impact by the number of research students, postdoctoral fellows, visiting scientists, and national and international collaborators (Fig. 6), the geographic spread (Fig. 7) and influence of the technique, the total number of research outputs that are attributable to a single scientist practicing a technique, the strength of coauthorship as an indicator of collaboration (Figs. 8–10), and the number of citations of individual research outputs (journal articles, invited review papers, or chapters) (Fig. 11). These are just a few of the indices that could be used to measure research impact. Similar initiatives have been applied to other bibliographical resources (10, 36, 51) and names elicited from social networks linked to newspaper content (29).

First and second generation fellows, students, visitors, and other associates of Dr. Burg. It should be noted that Dr. Jack Orloff mentored Dr. Burg while he was a fellow in the LKEM. Norica Green was a research associate with Dr. Burg for many years. Dr. James E. Bourdeau worked with Dr. Frank A. Carone and Dr. Charles E. Ganote for his PhD before working with Dr. Burg. Dr. C. Terrance Hawk and Dr. Delon W. Barfuss learned the perfused technique from Dr. Wiliam H. Dantzler before working with Dr. James A. Schafer. Dr. Lucia H. Kudo learned from Dr. Antonio Rocha, who learned from Dr. Juha P. Kokko. Dr. Yasuhiko Iino worked with Dr. Masashi Imai in Japan, and Dr. Imai was a fellow with Dr. Kokko. Dr. Michael F. Horster was on sabbatical leave with Dr. Burg (Knepper MA, personal communication) and later taught Dr. Rainer Greger the technique, and Dr. Greger taught Dr. Eberhardt Schlatter (Novak I, personal communication). Dr. Steve C. Hebert learned the technique from Susan Troutman Halm and Mary Lou Watkins, who were members of Dr. Schafer's laboratory.

Network of coauthorship of Dr. Burg with his first generation collaborators. The coauthorship data are based on journal articles (via PubMed searches; not invited review articles) in which an isolated perfused preparation was used.

Network of coauthorship of early (1966–1978) first generation collaborators with their second generation links. The coauthorship data are based on journal articles (via PubMed searches; not invited review articles) in which an isolated perfused preparation was used. It should be noted that there are links from K. R. Spring, M. Imai, and R. Balaban to coauthors presented in Fig. 10.

Network of coauthorship of later (1978–1990) first generation collaborators with their second generation links. The coauthorship data are based on journal articles (via PubMed searches; not invited review articles) in which an isolated perfused preparation was used. It should be noted that there are links from K. R. Spring, M. Imai, and R. Balaban to coauthors presented in Fig. 9.

Web of Science citations of isolated perfused tubule papers with Dr. Burg and/or his first generation collaborators as coauthors. These citation data are based on journal articles, invited review papers, or chapters in which an isolated perfused preparation was used. The authors were unable to locate any journal articles in which Drs. Almeida, Lutz, or Tune used the isolate perfused preparation after leaving the Burg laboratory.

Figures 6–11 facilitate the scientific visualization of network, geographic, and citation data relating to the isolated perfused tubule technique. Scientific visualization aims to provide “. . . new scientific insight through visual methods” (37). It also incorporates information visualization, which has a representation and analytic emphasis on qualitative information (46). A subset of both is geographic visualization, which relates to representation and visual analysis of spatial phenomena both through “traditional” maps and the current array of interactive digital media (46). The visualizations in this paper incorporate all three categories since they are fed by quantitative (number of papers authored, number of coauthor links, number of citations), qualitative (linkages between coauthors, and from supervisor to fellows, students, and visitors), and geographic (location of supervisors, fellows, students, and visitors over time) data.

To assemble the data needed for the visualizations, we started with data gathering via the results of searches on Google, Google Scholar, PubMed-NBCI (PubMed), and Web of Science (Web of Knowledge is used for data gathering by Mao) (36) to measure the impact of Dr. Burg's technique. As shown in Table 1, with simple internet searches using key words, one can find 337,000 hits (as of the time of writing this manuscript) are listed for “isolated perfused renal tubule” on Google, 57,500 for Google Scholar, 1,110 hits with PubMed, and 861 with Web of Science. It is obvious that more hits would be generated if we refined our search words. The original Burg et al. (6) paper has been cited 1,033 times in Google Scholar and 1,085 times in Web of Science.

Impact metrics from Google, Google Scholar, and PubMed, and Web of Science (“hits” as of 3/28/16)

The PubMed data were used more specifically along with any available curricula vitae, home webpages, and personal communications to create data sets centered on Dr. Burg and his first generation fellows, visitors, and students (Table 2). Such data include their role relative to Dr. Burg at NIH (which was extended to the second generation, the “fellows of fellows” of Dr. Burg, shown in Fig. 6), their time at NIH (relayed in Figs. 8–10, Table 2), where they came from and where they went to after NIH (shown in the Fig. 7 map, Table 2), and the number of papers published both with Dr. Burg and after leaving NIH. The latter tallies were based on PubMed data, where bibliographical details were mined for the number of papers (first author, too) and number of coauthor pairs for Dr. Burg, his first and second generation collaborators and any other coauthors (Figs. 8–10). The number of citations for each of the identified papers was extracted from Web of Science.

The data were converted to JavaScript Object Notation (JSON) format (www.json.org), a data interchange format in wide use. A JSON file was created for each of the six visualization figures and typically consisted of objects (scientists, papers) consisting of members (attributes) with their values. For example, a scientist object can have member values such as an ID number, name of scientist, number of papers, and number of first-author papers. A paper object can have a number of citations member value. Both scientist and paper objects appear in the visualizations as circular nodes. Since there are multiple objects, they are arranged in a list or array. The other significant aspect to the JSON file is an array of paired members that typically appear as connecting undirected linear links in the visualizations. Members are normally paired by their ID number, and the pair can have a member value itself (e.g., to record the number of coauthored papers between two scientists). So as an example, the seminal 1966 paper alone would be recorded as having four scientist nodes with six pairwise links connecting them all:

The JSON files were processed in JavaScript using the D3.js library (d3js.org). D3 (Data-Driven Documents) binds data to a Document Object Model (DOM), which can then be transformed through the data. This means that a variety of web-based standards (HTML, SVG, CSS) can be used seamlessly, and the visualizations can be viewed and interacted with a web browser such as Internet Explorer, Firefox, or Google Chrome. In this case, we use D3 to create SVG graph visualizations from the nodes and links, often used for related data of this kind (44). The node and link JSON data are processed so that the nodes are subjected to a repelling force if in the vicinity of other nodes (with the links having spring-like properties to enable the force to take affect), facilitating no overlap of nodes in the display and mitigating the amount of link crossings (Fruchterman and Reingold present a graph drawing model that simulates similar principles in Ref. 14). Then, to construct the visualizations as seen in this paper, nodes are manually placed, being removed from the influence of the force as they are picked up.

For displaying the data for Dr. Burg, his first generation fellows, students, and visitors, then his second generation fellows and students, a graph with a radial hierarchical layout is used. Dr. Burg name is at the center, with his first generation nodes arranged clockwise in chronological order on the inner ring (the exceptions are Dr. Burg's supervisor J. Orloff and technician N. Green) and second generation nodes arranged on the outer ring (Fig. 6). The links have been color coded according to the type of relationship of the “parent” node with the “child” node (which is stored with the links in the JSON file). The visualization shows the rapid expansion of degree of collaboration from the first to the second generation, mostly with fellows, with only a few first generation scientists training the majority of the second generation scientists.

The geographic diffusion map (Fig. 7) is a type of flow map cartographically (46) and needed multiple nodes per first generation collaborator, as these nodes represented the various universities and health institutes where they were based throughout their career. One node for each collaborator had a common location: NIH. The links connected sequential nodes for a single collaborator, with information on whether the link represents the career stage before NIH, or one or more career stage(s) after NIH (Fig. 7). In the visualization, these links were dashed before residence at NIH and solid post-NIH. Since many collaborators came from and returned to the same place, the links were directed and curved so that both stages could clearly be shown. The location nodes were placed according to the background SVG base map. This has been created with Natural Earth data (naturalearthdata.com) of world countries and the United States. It was originally projected in an azimuthal equidistant projection centered on NIH (Bethesda, MD, 77.097°W, 38.986°N). Because most of the knowledge diffusion has occurred within the United States, we wanted that area to be largest in the map. Therefore, an approximation to a logarithmic projection (23) was calculated by applying the following formula to each state and country area, based on its centroid and its distance relative to Bethesda:

where w = weight and d = distance.

This weight was then used to parameterize a density-equalizing cartogram calculated through the Gastner and Newman method (15) using ScapeToad (scapetoad.choros.ch). This use of weighting in cartograms to expand areas of interest was introduced by Carroll and Moore (9). The cartogram thus gives more “room” to the United States and less room to further-flung countries, spatially accommodating the diffusion nodes for Dr. Burg's fellows, students, and visitors (Fig. 7) The visualization emphasizes the technique's rapid diffusion, mostly within the United States, with a cluster in the northeastern states. Also notable is the international reach of knowledge diffusion with notable out-migration to Europe, South America, Asia, and Africa (Fig. 7).

The three coauthorship visualizations (Figs. 8–10) use proportionally sized circle nodes to convey the number of papers in which an author is named (number proportional to square root of circle radius) and proportional-width links for the number of times a pair of authors have been coauthors on a paper. Figure 8 covers papers of which Dr. Burg is an author. His node is central, with first generation collaborators (from Fig. 6) arranged on the outer ring and other coauthors in the space between. Figs. 9 and 10 covers papers not in Fig. 8, of which a first generation scientist is an author. Their nodes are central, with second generation collaborators (from Fig. 6) arranged on the outer ring and other coauthors in the space between. Due to the numbers of nodes and links from first to second generation, we have had to split the network. The first group contains first generation scientists and collaborators that were mainly active from 1966–1978 and the second group covers 1978–1990. Three scientists (R. Balaban, M. Imai, and K. R. Spring) are linked to first generation scientists in both groups and so are duplicated and placed on the outside of the outer circle (Figs. 9 and 10). For all three visualizations, an explicit time frame has been applied, reading clockwise and divided into quarters (Fig. 8 has a duration of 1966–1986). In all cases, attempts have been made to place researchers in the time quadrant in which they were mostly active. For the Burg network, prolific coauthors include Orloff, Grantham, Knepper, and Good, with some collaboration between fellows (Fig. 8). Once the scientists had left NIH, they largely published within their own networks, collectively creating very complex patterns. However, in the later time frame (Fig. 10), there was increased collaboration between Burg's fellows (e.g., Knepper, Good, and Star) with prolific publishers including Kokko and Schafer in the earlier time frame and Knepper and Good in the later. As with Burg and his fellows, there were a few high-frequency first generation-second generation coauthor partnerships [e.g., Kokko and Jacobson; Schafer and Andreoli; Schafer and Troutman; Knepper and Chou; Good, Watts III and George (an unusual group of three that regularly published together)].

The paper citation visualization (Fig. 11) uses proportionally sized circle nodes to convey the number of Web of Science citations a paper has (number proportional to the square root of the circle radius). Links are used to group papers associated with a particular author (out of Dr. Burg or one of his first generation collaborators) together, although having been rendered invisible in the visualization. The 1966 paper can be seen in the center, with >1,000 citations, and the plethora of papers that has stemmed from it can be clearly seen, a few with ∼500 citations but the majority with >50 citations. A power law relationship may link papers and number of citations, also scientists and the number of publications or coauthors. De Souza and Barbastefano (12) noted that authors and the number of links followed the power law, but further investigation would be needed for this data set. All of the visualizations make use of Bertin's (1) graphical variables to visually represent data: color, particularly hue (Fig. 6), line texture (Fig. 7), and size (Figs. 8–11).

Major Advances in Kidney Physiology Made with the Isolated Perfused Tubule Technique

Beyond the numeric research impact and metric analyses, we return to the importance of Dr. Burg's contribution to renal physiology by examining the “physiological impact” of the studies that used the isolated perfused tubule preparation. Many advances in renal physiology have been made with the micropuncture technique before the ability to “sneak a look” into the “black box” of the isolated perfused tubule. The isolated perfused tubule allowed a “physiological microscopic” level of investigation that could be pursued by scientists. Others have written about the advances made with the perfused tubule preparation; nonetheless, we bring their thoughts together here (3, 7, 26, 28, Grantham JJ, personal communication).

It was quickly discovered that the various segments of the nephron have much different physiological “jobs.” For instance, in the transcript of a Legacy interview (26), Dr. Burg was asked by Dr. Mark Knepper to highlight what he thought was one of the important discoveries made by using the isolated perfused tubule preparation. Dr. Burg stated that “. . . part of the tubule called . . . the proximal tubule and discovering that the transport characteristics of that part of the tubule differed depending on which portion of it you looked at. For example, the early portions transported glucose very well and rapidly, the last portions transported [para-aminohippuric acid] very rapidly and very easily.” In the quotation above, Dr. Burg (3) was referring to work conducted by Dr. Bruce Tune, a fellow while in his laboratory (Fig. 6), when they reported that glucose absorption occurred across the apical membrane of the proximal tubule cells (47) while para-aminohippuric acid was secreted across the basolateral membrane (48). We alluded to an issue of Kidney International that was devoted to the isolated perfused tubule preparation published in 1982, only 16 years after the Burg et al. (6) paper. Drs. Harry R. Jacobson and Juha P. Kokko (a fellow of Dr. Burg's) (Fig. 6) contributed the initial remarks for that issue along with manuscripts (27, 28, 32). They (28) provided a list of the “new developments” made with perfused tubule studies which is quoted here: “i) identification of active chloride transport in the thick ascending limb of Henle, ii) demonstration of intrinsic transport difference among diverse segments of the proximal tubule [(NB: which now applies to many segments of the nephron)], iii) description of the site and mechanism of action of various hormones and second messengers on salt and water transport, iv) proposal of a new model of the countercurrent multiplication system without active transport within the inner medulla, v) detailed description of divalent anion and cation transport in the various nephron segments, vi) significant advancement in understanding the mechanism of H+/HCO3− transport across segments of proximal and distal nephron, vii) furthering our understanding of organic anions and cation transport, viii) specific localization of the site of action of various diuretics, and ix) in vitro evaluation of nephronal adaptive response to in vivo manipulation such as uremia and changes in acid-base balance.” In addition to the discoveries listed above by Jacobson and Kokko, in 1974, another “surprising” finding was reported by Grantham and colleagues (20). They determined that the net secretion of para-aminohippuric acid was sufficient to overcome the absorption of NaCl in the S2 and S3 segments of the proximal tubule causing sustained net secretion of fluid into the tubule, a finding that had never been contemplated for the mammalian nephron. This finding led to the discovery of how fluid accumulates within the cysts of patients with polycystic kidney disease in response to cAMP-dependent chloride secretion. This is by no means an exhaustive list of the accomplishments, as 34 years of perfused tubule research has occurred since the Jacobson and Kokko list was compiled.

Some of the key papers that contributed to the list above were expanded upon by Dr. James A. Schafer (Fig. 6) (visited Dr. Burg while he was a postdoctoral fellow of Dr. Thomas E. Andreoli at Duke University) in his commentary that accompanied the republication of the 1966 paper in 1997 (7). Some of the examples noted by Dr. Schafer are revisited here with additional comments. Grantham and Burg (18) provided the initial evidence that the cortical collecting duct exhibited increased permeability to water via a vasopressin (cAMP)-dependent mechanism. Of course, their results and results from other groups came full circle with the Membrane-Shuttle hypothesis proposed by Wade and colleagues (52), and eventually in 1995, Knepper and coworkers (41) demonstrated that vasopressin increased the number of aquaporin -2 (AQP2) channels trafficked to the apical membrane of collecting duct cells and increased the osmotic water permeability, measured with isolated perfused tubules of collecting duct cells.

Aspects of the countercurrent multiplication system (CCMS) have been examined with the isolated perfused tubule preparation. Indeed, one of the more important developments using the isolated tubule technique was the description of the countercurrent multiplication system that took advantage of the differing transport characteristics of the various nephron segments. Dr. Kokko communicated to the authors that ‘How urine was concentrated and diluted was one of the “hottest and most controversial” topics when I went to work with Moe’ (Kokko JP, personal communication). In 1972, Dr. Kokko drew the first hypothesis on the importance of the descending loop of Henle in overall operation of the CCMS (31, 33). Dr. Kokko firmly believes that “without his [Burg's] technique, and our subsequent studies, Rector and I would never have had the data to put together our model that did not require active transport out of the thin ascending limb of Henle” (Kokko JP, personal communication). Also in the early 1970s, Burg and Green (4) and Rocha and Kokko (42, 43) examined Na+ and Cl− transport in the rabbit thick ascending limb of loop of Henle (TAL). These authors provided the first evidence that the TAL exhibited a very low permeability to water and actively reabsorbed NaCl by a yet to be described mechanism (protein). Subsequently, in the early 1980s, Greger and Schlatter (21, 22) provided evidence that NaCl transport by the TAL was via the electroneutral Na+/K+/2Cl− cotransporter (now known as NKCC2, SLC12A1). Burg et al. (5) had previously demonstrated that furosemide reduced the NaCl transport of the rabbit TAL. Of course, we now have a number of clinical molecules that target NKCC2 in the treatment of high blood pressure.

Finally, acid-base homeostasis is crucial for the survival of cells and, indeed, humans. McKinney and Burg (38) perfused the rabbit cortical collecting duct to determine the effects of dietary acid and base on transport function of this segment. They reported that the cortical collecting duct was capable of secreting H+ of HCO3− depending upon the dietary intake. We know that different types of intercalated cells (α, β) play specific roles in H+ of HCO3− transport of the cortical collecting duct. Good and colleagues (16) examined the NH4+ and HCO3− transport of the rat TAL. They determined there were two ways that the TAL contributed to acid-base regulation. First, they reported that NH4+ was reabsorbed into the TALs not by nonionic diffusion. Additionally, this reabsorption of NH4+ provided a mechanism by which the NH4+, produced and secreted in the proximal tubules, could be transported into the interstitial fluid and be available to the collecting duct. Second, they suggested that the cortical and medullary ascending limbs reabsorbed HCO3− against both a voltage and concentration gradient.

There are many more examples of contributions made to renal physiology from experiments conducted with the isolated perfused tubule preparation. The reader is directed to reviews by Burg (2, 3), Grantham et al. (19), and Burg and Knepper (8) for further progress with the isolated perfused tubule in the early years of the technique.

Concluding Remarks

In 2014, the NIH established the annual Maurice B. Burg Lecture in honor of Dr. Burg's accomplishments in renal research. This lecture takes place each year in the fall in Bethesda and is sponsored by the NHLBI. The inaugural lecturer was Dr. Jared Grantham.

It is difficult to accept that it has been 50 years since Drs. Burg, Grantham, Abramow, and Orloff published their landmark paper (6) in the American Journal of Physiology. Their paper provided a bridge between micropuncture and molecular biological characterizations of the nephron. The discoveries made using the isolated perfused tubule technique have confirmed and extended many known and newly identified concepts in renal physiology. Also, with the completion of the human genome project, renal pathophysiological research continues. Many readers are aware that the isolated perfused tubule technique has been adapted to examine other biological systems that have flourished over the last few decades. It is hoped that the research collaborations, migrational visualization, metrics indices, and citation analyses presented in this paper allow the reader to appreciate the overall research complexity and outputs that have been generated from just the first and second generation of scientists who have been associated with Dr. Burg. Of course, we are all waiting for the discoveries that the isolated perfused preparation technique will bring during the next 50 years.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.L.H. and A.B.M. provided conception and design of research; K.L.H. and A.B.M. analyzed data; K.L.H. and A.B.M. interpreted results of experiments; K.L.H. and A.B.M. prepared figures; K.L.H. and A.B.M. drafted manuscript; K.L.H. and A.B.M. edited and revised manuscript; K.L.H. and A.B.M. approved final version of manuscript.

ACKNOWLEDGMENTS

Many thanks to the following individuals for conversations or e-mails which included Moe Burg, Jared J. Grantham, Mark A. Knepper, James A. Schafer, Robert A. Star, David W. Good, George J. Schwartz, Charles S. Wingo, Juha P. Kokko, Jean-Yves LaPointe, Elie Hogan, Dwight McKinney, Roger G. O'Neil, Sue Nicholson, Ivana Novak, Gustavo Frindt, David G. Warnock, William H. Dantzler, Susan Troutman, and Dan Halm. Many thanks to Mark Knepper for answering numerous e-mails about details of accuracy about fellows/visitors at the National Institutes of Health and providing the photos of Moe and the isolated perfused tubule rig. The authors have tried to be very diligent in identifying second generation fellows/students who learned the isolated perfused tubule preparation from the first generation fellows/visitors, who directly learned the technique from Dr. Burg. We apologize if we have inadvertently missed any individuals (first or second generation) who should have been included in our analyses. We also apologize to those individuals around the world who set up isolated perfused technique rigs independently of visiting or having been a fellow of Dr. Burg's but were not included in this manuscript. We thank Holger Regenbrecht for suggesting the D3.js JavaScript library as a way of graphically representing data for Figs. 6–11. We thank Jared, Mark, Juha, Jim, and Moe for helpful comments on an earlier version of the manuscript. The authors express sincere thanks to Jared for his insightful details about his time in the Burg laboratory and once leaving the laboratory. Finally, words cannot express the thanks and gratitude that the entire scientific community has for Moe and his isolated perfused tubule technique, and we all celebrate the 50th anniversary of the 1966 publication in the American Journal of Physiology.